Methods for preparing factor X, activated factor X, inactivated factor X and inactivated factor Xa, and pharmaceutical compositions comprising same

ABSTRACT

Methods for preparing Factor X, activated Factor X, inactivated factor X and inactivated factor Xa, compositions comprising Factor X and Factor Xa, inactivated Factor X and inactivated Factor Xa and methods of medical treatment using Factor X, Factor Xa, activated Factor X and inactivated Factor Xa are disclosed. The preparation methods comprise a chromatography step using an immobilised metal ion affinity chromatography substrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/601,930, filed on Nov. 25, 2009, which is a U.S. National Phasepatent application of International Application Serial NumberPCT/GB2008/001810, filed on May 29, 2008, which is herein incorporatedby reference in its entirety, and which claims priority from GreatBritain Patent Application 0710321.1, filed on May 30, 2007, which isherein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods for preparing factor X,activated factor X (factor Xa), inactivated factor X and inactivatedfactor Xa, compositions comprising factor X, factor Xa, inactivatedfactor X or inactivated factor Xa which are suitable for pharmaceuticaluse, and use of factor X, factor Xa, inactivated factor X or inactivatedfactor Xa for the treatment of various medical conditions.

Factor X is a coagulation factor normally present in human blood. FactorX deficiency is a rare bleeding disorder which affects between 1 in500,000 and 1 in 1,000,000 of the population. It is characterised by atendency to excessive bleeding, similar to that caused by factor VIIIand factor IX deficiencies in haemophilia A and B respectively. Thereare currently no licensed treatments specifically for factor Xdeficiency anywhere in the world. In particular, there are no clinicalconcentrates of factor X currently available for use in the treatment offactor X deficiency. Instead, physicians have to rely on infusions ofplasma or of prothrombin complex concentrates (PCC). However, there area number of disadvantages accompanying use of plasma or PCC for thetreatment of factor X deficiency.

Plasma contains only a low concentration of factor X (approximately 1unit per mL), so a very large infusion volume is needed to achieve evena small increase in a patient's circulating level of factor X. Treatmentis constrained by the need to stop infusion before a fully-therapeuticdose has been delivered, to avoid volume overload causing osmoticimbalance and transfusion-related acute lung injury. Plasma alsorequires refrigerated or frozen storage which can restrict availabilityto the patient.

Prothrombin complex concentrates (PCC) contain some factor X, but thisis only a very minor component of the total protein present. PCCs arenot assayed or labelled for this indication which leads to highlyvariable dosing. Many of these older products are made from pooledplasma without the added safety margin of multiple virus inactivationsteps during manufacture. Most require refrigerated storage which, likeplasma, can restrict availability to the patient. As the mainconstituent of PCC is prothrombin, and PCCs carry a risk of beingpartially activated by the manufacturing process, there is a risk thatuse of PCC will result in a thrombotic side effect during treatment,which could be fatal. Although PCC contain more factor X than plasma inthe available volume, it is desirable to contain the therapeutic dose inas small a volume as possible, particularly as many of the treatedpatients are young children.

WO 89/05650 discloses a method for at least partially separating vitaminK-dependent blood clotting factors, including Factor X, from a mixturecontaining at least one such factor, for example a prothrombin complexconcentrate. The method comprises adsorption of the mixture onto a metalchelate chromatography column. However, factor X produced according tothe method in WO 89/05650 still contains significant amounts ofprothrombin, up to 40-50% by weight. The presence of prothrombin in aconcentrate of factor X is undesirable because prothrombin has a longercirculating half-life than factor X. If a patient is infused withtreatment containing both proteins, this can cause a disproportionateand cumulative increase in plasma prothrombin which may then unbalancethe haemostatic equilibrium in favour of thrombin generation and clotformation (haemostasis/thrombosis). Removal or reduction of prothrombinin a factor X concentrate would be desirable to minimise the risk ofspontaneous thrombin generation during the manufacture or storage of theproduct, which could cause also thrombotic reactions during clinicaluse.

There is therefore still a need for a process for the preparation offactor X which is efficient, can be carried out on an industrial scale,which allows incorporation of multiple virus reduction steps and whichprovides a pharmaceutically useful product. There is also a need forpharmaceutical formulations of factor X for use in the treatment ofconditions such as factor X deficiency.

SUMMARY

In one aspect, the invention therefore provides a method for theseparation of factor X from a starting material comprising factor X andprothrombin, the method comprising use of immobilised metal ion affinitychromatography (IMAC, also referred to as metal chelate chromatography).The method comprises:

-   -   a) adsorbing the starting material onto an immobilised metal ion        affinity chromatography substrate;    -   b) eluting selected adsorbed proteins from the substrate; and    -   c) monitoring the eluate for commencement of elution of        prothrombin and factor X, discarding a first portion of the        eluate and collecting a subsequent second portion of the eluate        enriched in factor X.

Preferably, the factor X is human factor X. Also preferably, the factorX obtained in step c) is further purified by anion exchangechromatography. Anion exchange chromatography can provide additionalseparation of factor X from prothrombin. In addition, it provides agentle method for concentrating the factor X to apharmaceutically-useful potency for formulation. Alternatively, thefactor X could be concentrated after step c) using a process such asultrafiltration. However, this may lead to greater mechanical loss andphysical damage to the factor X than does anion exchange chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an elution profile for factors X, II, and IX duringanion exchange chromatography according to one aspect of the presentdisclosure.

FIG. 2 illustrates a flowchart of an example process according to thepresent disclosure.

DETAILED DESCRIPTION

In the method disclosed in WO 89/05650, the prothrombin and the factor Xco-elute from the substrate, leading to factor X fractions which containhigh amounts of prothrombin. Surprisingly, it has now been found thatprothrombin is eluted preferentially at the front of the protein elutionpeak, whilst the factor X is eluted at a substantially constantconcentration throughout the protein elution. It is therefore possibleto exclude the main peak of protein elution, and collect factor X onlyin the tail part of the peak without significant loss of yield and withconsiderable increase in purity compared to the method disclosed in WO89/05650.

The starting material for the method of the invention may be anysolution comprising at least prothrombin and factor X. Preferably, thestarting material will be plasma or a plasma-derived fraction such ascryoprecipitate-depleted plasma and/or Fraction I-depleted plasma, morepreferably an extract from a plasma fraction. Most preferably, thestarting material is a mixture of prothrombin (factor II), factor IX andfactor X, for example prothrombin complex. Other proteins includingfactor VII, protein C, protein S, protein Z and inter-alpha-trypsininhibitor may also be present. Also preferably, the factor X will behuman factor X. Alternatively, the starting material could be a solutioncollected from a transgenic tissue or even from recombinant culturemedium from which a contaminating and co-fractionating protein such asprothrombin is also harvested.

The starting material may be prepared by any suitable method known inthe art. For example, methods for the separation of prothrombin complexfrom plasma and plasma fractions are well known in the plasmafractionation industry. Prothrombin complex is most commonly isolatedfrom plasma and plasma fractions (e.g. cryoprecipitate-depleted plasma,fraction I precipitate-depleted plasma or a mixture of cryoprecipitateand fraction I precipitate-depleted plasma) by anion-exchangechromatography. An alternative source of prothrombin complex-enrichedstarting material is the precipitate generated by ethanolicfractionation of plasma. In this process, different plasma proteins arepartitioned into different precipitate fractions by adjustment ofethanol concentration, temperature and pH. A particularly preferredstarting material is prothrombin complex obtained fromcryoprecipitate-depleted human plasma by anion exchange chromatographyusing an anion exchange medium such as DEAE-Sepharose (Feldman P A,Bradbury P I, Williams J D, Sims G E, McPhee J W, Pinnell M A, Harris L,Crombie G I, Evans D R, Large scale preparation and biochemicalcharacterization of a new high purity factor IX concentrate prepared bymetal chelate affinity chromatography, Blood Coagulation andFibrinolysis 5: 939-948 (1994)). This anion exchange chromatography stepcan be performed chromatographically in column-mode, in bulk batch mode,or in a surface mode where the anion exchanger is attached to an inertfilter or membrane support.

In step a) of the method of the invention, the starting material isloaded onto an IMAC substrate. Preferably, the substrate is present in acolumn for ease of processing. Any suitable metal ion may be used,including divalent metal ions such as copper, zinc or nickel, preferablycopper. Suitable immobilised metal ion affinity chromatographysubstrates for use in the process of the invention include thosedisclosed in WO89/05650, methacrylate gel with multi-substituted ligandson the side chain spacers (e.g. Fractogel EMD Chelate from Merck),methacrylate gel with single chelating groups on the spacer arm (e.g.Toyopearl Chelate from Tosoh Bioscience), pressure-stable polymer withan iminodiacetic acid chelating functional group (e.g. Profinity IMACfrom Bio-Rad Laboratories) and cross-linked agarose gel (e.g., chelatingSepharose FF from GE Healthcare). A preferred substrate is chelatingSepharose FF charged with copper ions.

Steps a) and b) of the method of the invention can be performedchromatographically in column-mode or in bulk batch mode. Alternatively,these steps may also be performed in a surface mode where the chelationcomplex is attached to an inert filter or membrane support. Columnchromatography is however preferred for ease of operation on anindustrial scale.

The loading conditions, including the buffer used, should be chosen suchthat the factor X (and co-binding prothrombin) in the starting materialare bound to the substrate. The salt concentration of the startingmaterial can be adjusted to minimize non-specific protein adsorption.Unwanted contaminants which do not bind or bind only weakly to thesubstrate may then be removed by washing. For example, if the startingmaterial has previously been subjected to a solvent-detergentvirus-inactivation step, most of the solvent or detergent reagentsremaining do not bind to the substrate and are easily removed bywashing. Any protein which is only weakly bound to the substrate mayalso be removed during the washing step. Alternatively, if the unwantedcontaminants bind to the substrate they may be removed by selectiveelution before the factor X is eluted, or they may remain bound to thesubstrate whilst the factor X is selectively eluted.

Loading and washing can be performed at relatively high ionic strength(e.g. 500 mM sodium chloride and 100 mM buffer salts) to minimisenon-specific binding of protein to the IMAC medium and to provideeffective removal of most unwanted chemicals which may be present in thestarting material. Inclusion of citrate in the buffer can remove some ofthe weakly-bound metal ions which may otherwise contaminatesubsequently-eluted proteins.

The eluate should be monitored for protein content, for example bymonitoring for absorbance at 280 nm. Absorbance at 280 nm is a measureof non-specific protein elution. Alternatively, the eluate could besampled and assayed for prothrombin and factor X using methods known inthe art. Initial washing should be continued until no more weakly boundprotein is being eluted. Prothrombin and factor X may then be elutedusing any suitable elution buffer. Elution of factor X may be achievedby reduction in the buffer ionic strength and/or change in the pH (e.g.from 6.5 to 7.0). Ionic strength reduction (e.g. from 500 mM NaCl to 100mM NaCl) and an increase in the pH of the buffer (e.g. from 6.5 to 7.0)are preferred in order to differentiate between the factor X and otherbound proteins.

Once protein elution is detected, the first portion of the eluatecollected should be discarded, as it will contain high levels ofprothrombin. Once the majority of the prothrombin has been eluted, asecond portion of eluate enriched in factor X (compared to the startingmaterial) should be collected. Once the elution behavior for aparticular IMAC substrate/starting material/elution buffer is known,then it may be possible to identify the first and second portions of theeluate simply on the basis of eluate volume. For example, if the IMACsubstrate is loaded into a column, then the first and second portions ofthe eluate may be defined by gel bed volume. In one non-limitingembodiment, the first portion of the eluate may comprise approximatelythe first 25% of the total eluate volume and the factor X-enrichedsecond portion may comprise the subsequent approximately 75% of thetotal eluate volume. Alternatively, the prothrombin and factor Xcontents of different eluate fractions can be measured using methodsknown in the art. For example, the first portion could be collecteduntil the ratio of prothrombin activity:factor X activity is less thanabout 0.01 unit/unit and the second, factor X-enriched portion after theratio of prothrombin activity:factor X activity has fallen below about0.01 unit/unit.

In a preferred embodiment, the second portion of the eluate enriched infactor X is further purified by anion exchange chromatography. It hassurprisingly been found that anion exchange chromatography can beeffective in removing residual prothrombin from the factor X, leading toan increase in purity of the factor X. It has been found thatprothrombin binds slightly more strongly than factor X to anion exchangechromatography media, with the result that it is eluted slightly laterthan factor X. At the chosen ionic strength for elution, the prothrombinelution peak also tends to be broader than the factor X elution peak.The overall effect is that prothrombin elution can be delayed relativeto factor X and the two can then be separated by selective collection ofthe eluate. The anion exchange chromatography step is also advantageousfor removing any residual metal from the metal chelate substrate whichmay be present in the eluate, and/or any residual solvent detergentreagents which may be present. Careful selection of buffer chemistryduring the anion exchange step allows for separation of factor X fromcontaminating prothrombin or factor IX (if present). The loading,washing and elution buffers for the anion exchange chromatography stepshould be chosen such that binding of the factor X to the media ismaximised, and the separation of factor X from contaminating prothrombinis also maximised.

Any suitable anion exchange chromatography medium may be used. Oneparticularly suitable medium is DEAE Sepharose anion exchange gel, forexample DEAE Sepharose Fast Flow.

Preferably, the anion exchanger is equilibrated with buffers containingonly citrate at about pH 6.0 before loading to maximise factor Xbinding. The loading conditions, including the buffer used, should bechosen such that factor X is bound to the medium. If necessary, thesecond portion of the eluate collected in step c) is diluted to reducethe ionic strength before loading onto the anion exchange chromatographymedium. A suitable conductivity is 10-18 mS/cm. There is a maximum saltconcentration/ionic strength beyond which factor X will not bind to theanion exchange chromatography medium. Preferably, the salt concentrationis adjusted to provide maximum binding in the minimum volume, so as toavoid excessive column loading/adsorption times, which could compromiseprotein integrity or microbiological safety.

After loading, washing with a suitable buffer removes any unboundimpurities, for example any residual solvent, detergent or metal whichmay be carried over from earlier processing steps. A preferred washbuffer contains both citrate and phosphate salts and raised sodiumchloride concentration to remove any residual chemical reagents usedduring previous stages of purification. Use of such a wash buffer alsoremoves citrate and phosphate anions which bind weakly to the anionexchange medium during equilibration and sample loading at low ionicstrength, but does not elute factor X. It was found that removal ofgel-bound citrate and phosphate ions at this stage prevented theirrelease from the anion exchanger during the subsequent ionic strengthstep which eluted the bound protein. If not addressed by the washbuffer, it was found that the bound citrate and phosphate ions would beremoved at the increased salt front produced by the subsequent elutionbuffer step, resulting in disruption of the salt (increased ionicstrength) boundary and distortion of the sharp protein elution. Thiscould potentially compromise both the factor X potency and itsseparation from other proteins.

Purified factor X is then eluted using a suitable elution buffer.Preferably, the constituents of the elution buffer are selected to elutefactor X at a concentration which allows subsequent processing withoutthe need for a separate concentration or diafiltration step. A preferredelution buffer contains approximately 10 mM citrate, 10 mM phosphate and310 mM sodium chloride at pH7.0. This combination of salt and pHadjustment was found to elute factor X in a sharp peak, providingconcentrated product which needed no further concentration beforeformulation to a pharmaceutical product.

After elution from the IMAC substrate, or from the anion exchange mediumif used, the purified factor X may be formulated for pharmaceutical use.Preferably, this will involve freeze drying of the factor X, optionallyin the presence of a suitable stabilizer, to give a factor X concentratewhich is suitable for reconstitution for pharmaceutical use. A freezedried factor X concentrate is preferred over a factor X solution as itwill be stable for storage at higher temperatures and for longer periodsthan would a solution. It also provides a stable product which can besafely exposed to a terminal heat-treatment virus-inactivation step.

One advantage of the process of the invention is that it allows easyincorporation of one or more virus inactivation or reduction steps.Suitable virus inactivation or reduction methods are known in the artand include pasteurisation, solvent detergent treatment, dry heattreatment and virus filtration. Preferably at least two virusinactivation or reduction steps are included in the method, morepreferably at least three.

For example, the starting material may be subjected to a virusinactivation step before it is loaded onto the immobilised metal ionaffinity chromatography substrate. Solvent detergent virus inactivationmay be carried out using reagents and methods known in the art (see forexample U.S. Pat. Nos. 4,481,189, 4,613,501 and 4,540,573, all of whichare hereby incorporated by reference). Suitable solvents includetri-n-butyl phosphate (TnBP) and ether, preferably TnBP. Suitabledetergents include polysorbate (Tween) 80, polysorbate (Tween) 20,sodium cholate and Triton X-100.

A preferred detergent is polysorbate 80 and a particularly preferredsolvent detergent combination is polysorbate 80 and TnBP.

A virus reduction step can be carried out on the eluate from either themetal chelate chromatography step or a subsequent anion exchangechromatography step. Filtration through a filter capable of removingviruses is preferred for the second virus reduction step. This includescommercially-available virus-retentive filters, for example the Planova15N virus-retentive filter (available from Asahi Kasei Medical, Japan),the Ultipor DV20 filter (available from Pall Corporation), the VirosartCPV filter (available from Sartorius) and the Viresolve filters(available from Millipore). A preferred filter is the Planova 15N filteras it provides good protein flux and virus removal. Such filters alsohave the potential to reduce or remove prions or other causative agentsof transmissible spongiform encephalopathy.

A virus inactivation step can also be carried out on the factor X afterit has been formulated. For example, a freeze dried formulation can besubjected to a terminal dry heat treatment virus inactivation step. Sucha step can involve heating the freeze dried formulation to about 80° C.for about 72 hours. These conditions are known to inactivate bothenveloped and non-enveloped viruses.

In a preferred embodiment, the method of the invention comprises threevirus reduction or inactivation steps: solvent detergent treatment ofthe starting material; virus filtration of the eluate from the metalchelate chromatography step or a subsequent anion exchangechromatography step; and dry heat treatment on the formulated finalproduct.

In a most preferred embodiment, the method of the invention comprises:

-   i) solvent detergent treatment of a starting material comprising    factor X and prothrombin;-   ii) adsorbing the solvent detergent treated starting material onto    an immobilized metal ion affinity chromatography substrate;-   iii) eluting adsorbed proteins from the substrate;-   iv) monitoring the eluate for commencement of elution of    prothrombin/factor X, discarding a first portion of the eluate and    collecting a subsequent second portion of the eluate enriched in    factor X;-   v) carrying out anion exchange chromatography to further purify the    second portion of the eluate;-   vi) filtering the product of step v) through a virus-retentive    filter;-   vii) freeze drying the filtered product of step vi), optionally in    the presence of one or more stabilizers; and-   viii) subjecting the freeze dried product to a virus    reduction/inactivation heat treatment step.

FIG. 2 illustrates a most preferred embodiment of the method of theinvention. The methods of the invention in one embodiment providefunctional factor X which has not been adventitiously activated (i.e.“native” factor X). Activation is undesirable for the treatment offactor X deficiency because activated factor X may cause unwantedthrombogenic side-effects. However, in another embodiment, the methodsof the invention may also be used to prepare activated factor X (factorXa) by incorporating an activation step into the method. Factor X can beactivated at any stage before formulation of the final product. Theactivation of factor X to factor Xa can be carried out at any stageduring the methods of the invention, including:

-   on the starting material, for example the source plasma, a plasma    fraction, or prothrombin complex;-   on the factor X enriched eluate from the immobilised metal ion    affinity chromatography step;-   on diluted factor X before loading onto an anion exchanger for    further purification;-   on factor X eluted from an anion exchanger; and on virus-filtered    factor X.

Preferably, the activation is carried out at some stage after theimmobilised metal ion affinity chromatography step, because anythingearlier might activate any other clotting factors present in thestarting material and compromise their suitability for manufacture intoother therapeutic products. Ideally, the activation step is carried outas late as possible in the manufacturing process, as once activated, thefactor X may be more labile (i.e. less stable). Also ideally, theactivation step is carried out at a stage in the method which allows theactivated factor X to be stored pending analysis before finalformulation, filling and freeze-drying. Most preferably, activationtakes place between the anion exchange step and formulation of thevirus-filtered factor X.

Activation of factor X to factor Xa can be carried out using anysuitable method. For example, factor X can be activated by calciumalone, or in combination with a venom such as Russell's Viper Venom.Other metal ions, particularly divalent metal ions such as magnesium,can also potentiate the activation of factor X. To minimize any risk ofactivation chemicals remaining in the final product, the activator canbe immobilised on a solid matrix and a factor X solution passed throughthe matrix. Activation occurs during the passage and the factor X flowsthrough without dilution. For example, metal ions may be immobilised ona chelating matrix. Alternatively, activation with metal ions can bedone in solution and the solution passed through a chelating matrix toremove the metal ions afterwards. Venoms such a Russell's Viper Venomcan be immobilised on ligand-activated matrices such as CNBr-activatedSepharose or epoxy-activated Sepharose.

At earlier stages in the factor X purification process, it may bepossible to use the feedback mechanism whereby residual prothrombin isconverted to thrombin by small amounts of activated factor X and thethrombin then accelerates the activation of more factor X. Factor X mayalternatively be activated by a temperature drop in the presence offactor VII thus utilising the “cold-activation” of factor VII, with thefactor VII then activating factor X.

The invention also provides a pharmaceutical composition comprisingfactor X or factor Xa and one or more pharmaceutically acceptablediluents, excipients and/or stabilizers. Preferably, the factor X orfactor Xa is factor X or Xa prepared by the method of the invention,although any factor X or factor Xa of suitable purity may be used.Factor X or factor Xa is preferably at least 30% by weight, morepreferably at least 70% by weight of the total protein present in anypharmaceutical composition, and most preferably is the only proteinpresent. The composition may be a frozen solution, in liquid form orfreeze dried, but is preferably freeze dried in order to maximise theshelf life. Freeze dried compositions are also preferable as they may besubjected to a dry heat treatment step for the inactivation of viruses.

Suitable stabilizers are compounds which help stabilize the factor X orfactor Xa during freeze drying and/or heat treatment steps (i.e. helpmaintain factor X or factor Xa activity across these steps). Dependingon the clinical indication for use of the composition, such stabilizersinclude, but are not limited to, carbohydrates such as sucrose,trehalose and dextran; amino acids such as glycine;polyvinylpyrrolidone; and polyethylene glycols. A preferred stabilizeris sucrose.

For a factor X or factor Xa solution for infusion, a pH between about6.0 and about 8.0 and a salt concentration between about 150 mM andabout 400 mM sodium chloride would be acceptable. Suitable buffersinclude those with citrate and phosphate concentrations between 5 mM and50 mM, and sodium chloride concentrations between 50 mM and 400 mM. Apreferred freeze dried composition comprises, on reconstitution, 1-2%w/w sucrose and 150-300 mM sodium chloride at a pH of 6.5-7.3. A morepreferred formulation comprises 1% w/w sucrose, 10 mM citrate, 10 mMphosphate and 200-300 mM sodium chloride at a pH of 7.3.

The amount of factor X in the final product should be such that, onreconstitution, it can provide a solution containing between about 20and about 200 international units of factor X per mL (or an equivalentamount of factor Xa when required for particular clinical applications).The desired concentration will be mainly governed by clinical,pharmaceutical and commercial considerations.

The freeze dried compositions of the invention can be prepared by addinga stabilizer and any additional formulation ingredients to a solution offactor X or factor Xa. The solution may optionally be sterile filtered,for example through a 0.2 μ m pore size filter, before being filled intosuitable containers, for example glass vials. The containers are thenfreeze dried. Suitable freeze drying conditions are freezing at about-50° C.; primary drying for about 72 to 98 hours at a shelf temperatureof about -20 to -34° C., preferably about -25 oC, and a chamber pressureof about 17 to 200 μ bar, preferably about 100 μ bar; and secondarydrying for about 18 hours at a shelf temperature of about +28 to +32°C., preferably +30° C. and a chamber pressure of about 0 to 60 μ bar,preferably about 30 μ bar. At the end of secondary freeze-drying, thecontainers are closed under vacuum and oversealed.

The composition ingredients are preferably selected such that thecomposition is stable over prolonged storage times, preferably 2-3years, can be stored at room temperature, and can be readilyreconstituted to give a factor X or factor Xa solution suitable forclinical use. Reconstitution of a freeze dried composition may becarried out by introducing sterile water for injections into the vial byan aseptic method. In practice, this involves the transfer of sterilewater into the vial using a needle and syringe or a commercial devicefor transferring pharmaceutical solvents. The freeze-drying processleaves the product in the vial under partial vacuum, so water is drawninto the vial by the vacuum, which can help the reconstitution process.The quicker the reconstitution the better, as the product could beneeded by a patient experiencing a critical bleed. A reconstitution timeof about 1-10 minutes is acceptable, but preferably the reconstitutiontime is less than about 60 seconds, and more preferably less than about30 seconds.

The freeze dried factor X or factor Xa concentrate can be subjected to adry heat

treatment virus reduction step. Suitable conditions include heatingbetween 60° C. and 100° C. for between 1 and 144 hours. Generally, lowertemperatures and/or shorter treatment times will result in higher yieldsof functional protein but less efficient virus inactivation. Thepreferred conditions are heat-treatment at about 80° C. for about 72hours as this has been found to provide both good virus inactivation andacceptable yield of factor X.

Factor X or factor Xa prepared according to the method of the inventionand the factor X or factor Xa compositions of the invention may be usedfor the treatment of factor X deficiency, and any other conditions forwhich treatment with factor X has been proposed. In particular, themethod of the invention allows the preparation of highly-purified factorX or factor Xa with multiple virus inactivation steps and which can bepresented to the patient in a small volume, concentrated formulation.The formulations of the invention also provide a stable freeze-driedproduct which can be stored for extended periods at ambient or elevatedtemperatures.

Factor X, including that prepared according to the method of theinvention and the factor X compositions of the invention, may also beused for the treatment of patients who have developed inhibitors toblood clotting proteins. Some patients (about 5-10%) with factor VIII orfactor IX deficiency (Haemophilia A or B) develop inhibitors orautoantibodies in response to replacement therapy with concentrates offactor VIII or factor IX. These patients are extremely difficult totreat during acute bleeding episodes which can be life-threatening.Factor X concentrate and/or activated factor X (factor Xa) concentratecould be used to treat these patients by stopping acute bleedingepisodes.

Factor X acts directly at the penultimate stage of the blood clottingreaction process. Factor X is activated and then catalyses theactivation of prothrombin to thrombin which in turn cleaves fibrinogenand factor XIII to form the cross-linked fibrin clot. Factor VIII is acofactor in the activation of factor X which is catalyzed by activatedfactor IX. Thus deficiency of either factor VIII or factor IX could beby-passed by supplementation therapy with factor X or activated factor X(i.e. factor Xa). The rationale for efficacy of factor X concentrate isthat by raising the plasma concentration of factor X, the reactionequilibrium would move toward the endogenous generation of factor Xa,thereby driving the clotting cascade reaction towards clot formation.The rationale for efficacy of factor Xa concentrate is that the thrombingeneration from prothrombin would be driven by the natural catalyst(factor Xa) albeit by infusion rather than reliance on the earlierstages of coagulation pathway activation.

The use of factor X or factor Xa to treat patients with factor VIII orfactor IX deficiency forms a further feature of the invention. Thus, theinvention also provides a method of treating factor VIII or factor IXdeficiency in a patient, the method comprising treating the patient witha therapeutically effective amount of factor X or factor Xa. Theinvention also provides factor X or factor Xa for use in treating factorVIII or factor IX deficiency in a patient.

Factor X or factor Xa, including that prepared according to the methodof the invention and the factor X or factor Xa compositions of theinvention, may also be used as a haemostatic aid for patients who do nothave factor X deficiency but who are suffering from haemorrhage or therisk of haemorrhage. Treatment with factor X or activated factor X(factor Xa) may serve to correct the haemostatic imbalance, thuspromoting coagulation.

The use of factor X or factor Xa to treat patients suffering fromhaemorrhage or the risk of haemorrhage forms a further feature of theinvention. Thus, the invention also provides a method of treating orpreventing haemorrhage in a patient, the method comprising treating thepatient with a therapeutically effective amount of factor X or factorXa. The invention also provides factor X or factor Xa for use intreating or preventing haemorrhage.

Factor X, including that prepared according to the method of theinvention and the factor X compositions of the invention, may also beused for anticoagulant reversal. Some anticoagulants work by depletingthe plasma of functional clotting factors. Sometimes it is necessary toreverse this effect as quickly as possible, for example to allowemergency surgery. This is currently achieved by injection ofprothrombin complex concentrate (PCC) which is a mixture of clottingfactors. However, as the balance of clotting factors in PCC does notmatch the balance in anticoagulated patients, there can be hazardsassociated with loss of haemostatic control when the PCC is infused. Useof factor X concentrate, possibly alongside other purified clottingfactors, may provide better clotting control in a patient.

The use of factor X or factor Xa for anticoagulant reversal forms afurther feature of the invention. Thus, the invention also provides amethod of reversing the effect of an anticoagulant in a patient, themethod comprising treating the patient with a therapeutically effectiveamount of factor X or factor Xa. The invention also provides factor X orfactor Xa for use in anticoagulation reversal.

Inactivated factor X may be used for the treatment of patients with athrombotic tendency. Some patients have a thrombotic tendency orhypercoagulable condition which can be treated with anticoagulants.Historically the choice of treatment has been limited to warfarin andits analogues (which inhibit the carboxylation of clot-forming VitaminK-dependent clotting factors) or heparin and its analogues (whichcatalyse the inhibition of activated clotting factors by proteaseinhibitors). Some conditions (e.g. pregnancy or surgery) do not favourthe use of these therapies, either because of concern about toxicity orbecause they complicate the rapid measurement and control of acutebleeding problems. Treatment with an inactivated factor X concentratecould reduce the thrombotic tendency by anticoagulant action. Theadvantage of treatment with a purified inactivated factor X concentrateis that there would be very little other protein present which couldcause unwanted side-effects.

The rationale for this treatment is that a suitably inactivated factor Xwould have no catalytic capability to convert prothrombin to thrombin(due to denaturation of the factor X active site) but would retaincapability to bind to the natural substrates in blood. The inactivatedfactor X would compete for these sites with the endogenous active factorX. If administered at an appropriate dose, the inactivated factor Xcould saturate the substrate sites, down-regulating the clotting system.

The use of inactivated factor X or inactivated factor Xa as ananticoagulant forms a further feature of the invention. Thus, theinvention also provides a method of treating or preventing coagulationin a patient (e.g. a thrombotic or hypercoagulable state), the methodcomprising treating the patient with a therapeutically effective amountof inactivated factor X or inactivated factor Xa. The invention alsoprovides inactivated factor X or inactivated factor Xa for use as ananticoagulant, and pharmaceutical compositions comprising inactivatedfactor X or inactivated factor Xa and one or more pharmaceuticallyacceptable diluents, excipients and/or stabilizers.

Inactivated factor X or inactivated factor Xa is preferably at least 30%by weight, more preferably at least 70% by weight of the total proteinpresent in any pharmaceutical composition, and most preferably is theonly protein present. The composition may be a frozen solution, inliquid form or freeze dried, but is preferably freeze dried in order tomaximise the shelf life. Freeze dried compositions are also preferableas they may be subjected to a dry heat treatment step for theinactivation of viruses.

Any method of inactivation which causes denaturation of the factor Xactive site without destroying the capability of the factor X to bind tonatural substrates in blood can be used to inactivate the factor X orfactor Xa. For example, factor X (or factor Xa) can be inactivated byheat (e.g. at 60° C. in solution for >20 hours, or at 80° C. for 72hours after lyophilisation in a formulation which renders the factor Xmore susceptible to inactivation, e.g. a formulation containingpolyvinyl pyrrolidone, or polyethylene glycol, of low ionic strengthand/or of high sugar content), by exposure to a source of gammairradiation, or by reaction with denaturing or cross-linking chemicals.Inactivation can be verified by assaying for factor X activity beforeand after the treatment. Any factor X or factor Xa could be inactivatedfor use as an anticoagulant. Preferably, the factor X or factor Xa wouldbe prepared according to the method of the invention or would be afactor X or factor Xa composition according to the invention. Thus, inanother aspect, the methods of the invention further comprise aninactivation step to inactivate factor X or factor Xa.

Administration of factor X, factor Xa, inactivated factor X orinactivated factor Xa to a patient will normally be via intravenousadministration of a suitable solution. Other routes of administration(e.g. subcutaneous, oral) may be used in the presence of a suitablecarrier, but clinical response may be delayed and/or reduced and thusintravenous administration is preferred. Suitable dosage regimens willdepend on the patient and condition being treated and on the mode ofadministration. In chronic conditions such as factor X deficiency,repeated treatments will be necessary. For other conditions, a singletreatment may be sufficient.

Any and all possible combinations of preferred features disclosed hereinform part of the invention, even if such combinations are not explicitlydisclosed.

The following assay methods may be used to measure protein activity inthe methods and compositions of the invention.

Clotting Assay for Factor X

Factor X can be measured in a clotting assay which uses factorX-deficient plasma as the substrate. Test material is added to theplasma at predetermined dilution(s) and the clotting time of the plasmaafter re-calcification is measured. The clotting time is then comparedto the clotting times obtained from dilutions of a factor X standardisedpreparation (e.g. the WHO International Standard for factors II, IX andX) and the concentration of factor X is interpolated.

Chromogenic Assay for Factor X

Factor X can be measured by first activating the factor X with Russell'sViper venom factor X specific activator and then determining theincrease in absorbance due to release of a chromophore from a commercialchromogenic peptide substrate (e.g. Chromogenix S2765). This is thencompared to the absorbance change obtained from dilutions of a factor Xstandardised preparation (e.g. the WHO International Standard forfactors II, IX and X) and the concentration of factor X is interpolated.

Antigenic Assay for Factor X (ELISA)

Factor X antigen can be measured by enzyme linked immunosorbant assaywith antibodies specific to factor X, one of which is conjugated to aperoxidase enzyme. Addition of a peroxidase specific substrate resultsin the release of a chromophore. This is then compared to the absorbancechange obtained from dilutions of a factor X standardised preparation(e.g. the WHO International Standard for factors II, IX and X) and theconcentration of factor X is interpolated.

Chromogenic Assay for Prothrombin

Prothrombin can be measured by first activating the prothrombin withEcarin and then determining the increase in absorbance due to release ofa chromophore from a commercial chromogenic peptide substrate (e.g.Chromogenix S2238). This is then compared to the absorbance changeobtained from dilutions of a prothrombin standardised preparation (e.g.the WHO International Standard for factors II, IX and X) and theconcentration of prothrombin is interpolated.

Protein Assay

The Pierce BCA assay combines the well known reduction of Cu²⁺ to Cu⁺ byprotein in an alkaline medium (biuret reaction) with the highlysensitive and selective colormetric detection of the Cu⁺ using a uniquereagent containing bicinchoninic acid.

The invention is further illustrated in the following non-limitingexamples.

EXAMPLE 1 Separation of Prothrombin from Factor X Using Metal ChelateChromatography

Prothrombin complex was applied to a column of chelating Sepharose whichhad been charged with copper ions. After washing with 50 mM citrate 50mM phosphate 500 mM sodium chloride buffer at pH 6.5, factor X wasrecovered by elution with buffer containing 50 mM citrate 50 mMphosphate 100 mM sodium chloride at pH 7.0. Consecutive equal fractionswere collected from the first observed rise in eluate absorbance at 280nm until the return to baseline. Factor X and prothrombin were measuredin each fraction. Results are shown in Table 1-1.

TABLE 1-1 Factor X and Prothrombin 5 elution from metal chelatechromatography Prothrombin, Factor X, Prothrombin, iu per unit Fractioniu/mL iu/mL of factor X 1 17.3 0.49 0.028 2 19.7 0.37 0.019 3 18.7 0.220.012 4 21.1 0.17 0.008 5 19.7 0.13 0.006 6 22 0.10 0.005 7 18.4 0.080.004 8 17.4 0.07 0.004 9 16.2 0.06 0.004 10 16 0.06 0.003 11 13.6 0.0050.004 12 13.7 0.05 0.003 13 12.8 0.04 0.003

This Example demonstrates that whilst the factor X content of eachfraction was reasonably constant, the prothrombin content fellsignificantly after the first few fractions.

EXAMPLE 2 Further Purification of Factor X by Anion ExchangeChromatography

Factor X-rich eluate from metal chelate chromatography was diluted toreduce ionic strength then applied to a column of DEAE-Sepharose, whichhad been packed and equilibrated in citrate phosphate buffer. The loadedcolumn was washed with the same equilibration buffer and then elutedwith citrate-phosphate buffer containing sodium chloride. The eluate wascollected and assayed for factor X and prothrombin. The different buffercompositions are shown in Table 2-1 and results are shown in Table 2.2.These show how buffer conditions can be modified to remove prothrombinfrom factor X, reduction in phosphate and pH both being preferredembodiments of the invention.

TABLE 2-1 Buffer compositions used for different runs Run CitratePhosphate NaC1 pH Equilibration Buffer, mM A 10 0 100 6.0 B 10 10 1007.0 C 10 0 100 6.0 D 10 0 100 6.0 Elution Buffer, mM A 10 10 310 7.0 B10 10 310 7.0 C 12 0 310 6.5 D 10 10 310 7.0

TABLE 2.2 Separation of factor X from prothrombin using different bufferformulations Eluate Prothrombin, Factor X, Prothrombin, iu per factorProthrombin Run iu/mL iu/mL X iu reduction factor A 204 0.075 0.00037 23-fold B 673 2.35 0.0035 2.4-fold C 162 0.034 0.0021  40-fold D 2590.50 0.0019 2.9-fold

EXAMPLE 3 Improved Removal of Prothrombin by Metal ChelateChromatography

Solvent-detergent treated prothrombin complex was applied to a column ofchelating Sepharose which had been charged with copper ions. Afterwashing with 50 mM citrate 50 mM phosphate 500 mM sodium chloride bufferpH 6.5, factor X was recovered by elution with buffer containing 50 mMcitrate 50 mM phosphate 100 mM sodium chloride buffer pH 7.0. Factor Xwas collected either from when the eluate absorbance at 280 nm startedto increase until when it had returned to baseline (Method A, asdescribed in WO 89/05650) or for a quantity of 3.3 gel bed 15 volumesstarting 1.2 gel bed volumes after the rise in eluate absorbance (MethodB according to the invention). Results are shown in Table 3-1.

TABLE 3-1 Mean composition of metal chelate eluate collected bydifferent methods Prothrombin, Factor X, Prothrombin, iu per iu ofProthrombin Method iu/mL iu/mL factor X removal factor A (n = 7) 15.90.42 0.026 — B (n + 6) 16.8 0.12 0.007 3.7-fold

EXAMPLE 4 Further Purification and Formulation of Factor X to YieldActive and Nonactive Products

Factor X was prepared by purification of solvent-detergent treatedprothrombin complex using the metal chelate chromatography method of theinvention. The factor X was then applied to a column of DEAE SepharoseFF equilibrated in citrate buffer containing 100 mM sodium chloride atpH6.0. The factor X was eluted as a single peak using acitrate-phosphate buffer containing 310 mM sodium chloride at pH7.0.

The factor X eluate was formulated with buffer containing differentpotential stabilizers, dispensed into glass vials and freeze-dried. Oncompletion of freeze-drying, the vials were sealed and then subjected tothe virus-inactivation process of heating at 80° C. for 72 hours. Thestart material and product were then reconstituted and assayed.Reconstitution time, solution appearance and factor X yield weremeasured. Factor X yield is evidence of the retained protein capacityfor activation (high yield) or the amount of proteininactivation/denaturation (low yield). Table 4-1 shows formulationswhich resulted in good reconstitution properties and yield of factor Xactivity. Table 4-2 shows formulations which resulted in factor Xinactivation (low yield), suitable for the preparation of an inactivatedfactor X product.

Reconstitution Solution Factor X Formulation time, seconds Appearanceyield, % 10 mM citrate, 10 mM <25 Clear 74 Phosphate, 150 mM SodiumChloride pH 7.3 + 0.5% sucrose 10 mM citrate, 10 mM <20 Clear 88Phosphate, 150 mM Sodium Chloride pH 7.3 + 1% sucrose 10 mM citrate, 10mM <20 Clear 70 Phosphate, 210 mM Sodium Chloride pH 6.5 + 0.5% sucrose10 mM citrate, 10 mM <20 Clear 78 Phosphate, 250 mM Sodium Chloride pH6.8 + 2% sucrose 10 mM citrate, 10 mM 25 Clear 84 Phosphate, 250 mMSodium Chloride pH 7.0 + 1% sucrose 10 mM citrate, 10 mM <20 Clear 84Phosphate, 250 mM Sodium Chloride pH 7.3 + 2% sucrose 10 mM citrate, 10mM <25 Clear 74 Phosphate, 300 mM Sodium Chloride pH 7.3 + 0.5% sucrose10 mM citrate, 10 mM <20 Clear 73 Phosphate, 300 mM Sodium Chloride pH7.3 + 3% sucrose 10 mM citrate, 10 mM <20 Clear 77 Phosphate, 400 mMSodium Chloride pH 7.3 + 1% sucrose 10 mM citrate, 10 mM <25 Clear 81Phosphate, 400 mM Sodium Chloride pH 7.3 + 1.5% sucrose 10 mM citrate,10 mM <20 Clear 92 Phosphate, 300 mM Sodium Chloride pH 7.3 + 1%trehalose 10 mM citrate, 10 mM <20 Clear 82 Phosphate, 300 mM SodiumChloride pH 7.3 + 3% trehalose 10 mM citrate, 10 mM <25 Clear 72Phosphate, 270 mM Sodium Chloride pH 6.0 + 1% sucrose, 1% Dextran35-45K. 10 mM citrate, 10 mM <20 Clear 52 Phosphate, 250 mM SodiumChloride pH 7.0 + 20 mM glycine

TABLE 4-2 Formulations resulting in factor X inactivation SolutionFactor X Formulation Reconstitution Appearance yeild, % 10 mm citrate,45 Clear 1.6 10 mm Phosphate, 240 mm Sodium Chloride ph 6.5 + 3% SUCROSE10 mM citrate, >300 Clear 0 10 mM Phosphate, 200 mM Sodium Chloride pH6.5 + 3% SUCROSE 10 mM citrate, >300 Clear 0.8 10 mM Phosphate, 200 mMSodium Chloride pH 7.3 + 3% sucrose 10 mM citrate, >300 Clear 0.8 10 mMPhosphate, 200 mM Sodium Chloride pH 6.8 + 3% sucrose 110 mMcitrate, >600 Coloured 0.8 10 mM Phosphate, Precipitate 250 mM SodiumChloride pH 7.0 + 1% sorbitol 10 mM citrate, 48 Clear 13 10 mMPhosphate, 250 mM Sodium Chloride pH 7.0 + 1% PVP 40K 10 mM citrate, >20Clear 27 10 mM Phosphate, 250 mM Sodium Chloride pH 7.0 + 1% PEG 4000(In Table 4-2, PVP = polyvinyl pyrrolidone, and PEG = polyethyleneglycol).

EXAMPLE 5 Demonstration that Inactivated Factor X Will Inhibit theCoagulation Process

Factor X prepared by the method of the invention was inactivated byheating in solution at 60° C. for >20 hours. Inactivation was verifiedby assaying for factor X activity before and after the treatment.

Two standard clinical laboratory coagulation tests were used todetermine the effect of adding the inactivated factor X to normal pooledplasma. 50 μl of inactivated factor X was mixed with 50 μl of pooledhuman normal plasma. 50 μl of fully-functional factor X or factor Xformulation buffer were substituted for the inactivated factor X in sometests to serve as positive and negative control respectively. As afurther control, additional calcium was used in one set of tests toovercome possible calcium-chelation by citrate ions in the factor Xformulation buffer. The samples were then assayed in the prothrombintime (PT) test or the activated partial thromboplastin time (APTT) test.These two tests are well established in haematology and coagulationlaboratories where they are used to distinguish between clinical defectsin the extrinsic or intrinsic coagulation mechanisms.

PT method: 50 μl of test sample and 50 μl of plasma were incubated for60 seconds then 100 μl Stago Neoplastine (rabbit brainthromboplastin+Calcium chloride) was added. The clotting time wasmeasured.

APTT method: 50 μl of test sample and 50 μl of plasma were mixed with 50μl DAPTTIN (kaolin, sulphatide+highly purified phospholipid) andincubated for 180 seconds. 100 ul 25 mM calcium chloride was added andthe clotting time was measured.

Results are shown in Tables 5-1 and 5-2.

TABLE 5-1 Prothrombin Time Factor X deactivated at Factor X at 100 iu/mlin 100 iu/ml in Assay formulation buffer formulation buffer buffer (sec)(sec) (sec) No sample 34.7 21.6 15.2 dilution 1:10 dilution 16.3 15.51:100 dilution 13.8 15.0 Added 10 mM CaC1₂: No sample 32.9 17.6 dilution1:10 dilution 16.9 14.5 1:100 dilution 14.2 14.6

TABLE 5-2 Activated Partial Thromboplastin Time Factor X deactivated atFactor X at 100 iu/ml in 100 iu/ml in formulation formulationFormulation Assay buffer buffer Buffer buffer (sec) (sec) (sec) (sec) Nosample 70.3 41.2 37.7 33.0 dilution 1:10 33.1 31.3 31.5 dilution 1:10032.1 30.7 33.9 dilution Added 10 mM CaC1₂: No sample 60.5 22.5 dilution1:10 35.8 31.9 dilution 1:100 32 32.1 dilution

These results show that both the prothrombin time and the activatedpartial thromboplastin time were prolonged by addition of theinactivated factor X in a dose-dependent manner. The effect wasattributable to inactivated factor X. No effect was seen on the PT withnative, active factor X and only a much smaller effect was seen on theAPTT with native, active factor X. Buffers or salts had no effect ineither test.

EXAMPLE 6 Further Purification of Factor X by Anion ExchangeChromatography

Factor X which had been eluted from a copper-charged, prothrombincomplexloaded IMAC column was diluted to a conductivity of 15 mS/cm atpH6.0. It was applied to a pre-packed column of DEAE-Sepharose Fast Flowanion exchanger. The loaded column was then washed with gelequilibration buffer (approx 10.5 mM citrate 100 mM sodium chloridepH6.0). Buffer composition was then adjusted by washing withapproximately 10 mM citrate 10 mM phosphate 150 mM sodium chloride pH6.0before eluting the bound protein with approximately 10 mM citrate 10 mMphosphate 310 mM sodium chloride pH7.0. Fractions were collected acrossthe protein elution peak and assayed for factors X, II and IX. Resultswere expressed as a percentage of the maximum activity (in iu/mL) foreach protein (FIG. 1). Results showed that factor II and factor IXelution is retarded by this anion exchange step. The step may be used toseparate factor X from factor II and factor IX by selective fractioncollection.

EXAMPLE 7 Activation of Factor X and its Use to By-Pass Factor VIIIFunction

Factor X eluate produced by sequential anion exchange chromatography ofcryoprecipitate-depleted plasma, chromatography on copper-chargedchelating gel and further chromatography on anion exchanger wasactivated. Activation was achieved by incubation of the factor X-richeluate with 25 mM calcium chloride and 0.01 u/mL of Russell's vipervenom-X at pH7.4 at 37° C. Activated factor X was measured using achromogenic substrate assay for factor X without the addition of assayactivator. Activity was determined by interpolation from a standardcalibration line generated with a standard for factor X and the normalassay activator.

The activated factor X was tested in a kaolin-activated partialthromboplastin time assay using factor VIII-deficient plasma as thesubstrate (50 μl factor VIIIdeficient plasma+50 μl assay buffer+50 μldapttin TC reagent incubated at 37° C. for 180 seconds before additionof 100 μl of the test sample). The clotting times were measured andcompared with those for control samples which contained only the factorX activators or non-activated factor X. Results shown in Table 7-1demonstrate that (a) factor X can be activated by calcium chloride or bya combination of calcium chloride and RVV-X; (b) the activated factor Xsubstantially shortens the clotting time and this shortening is greaterthan achieved by any of the equivalent factor X activators alone.

TABLE 7-1 Use of activated factor X as a Factor VIII by-pass ClottingInitiator (components reacted Mean FVIII-deficient together for 30minutes at 37° C. before APTT clotting time addition to APTT substrate)(seconds) 25 mM calcium chloride 107.0 10 iu/ml factor X + 25 mM calcium6.7 chloride + 0.01 u/ml RVV-X 10 iu/ml factor X + 25 mM calcium 60.7chloride 25 mM calcium chloride + 0.01 u/ml 23.7 RVV-X

EXAMPLE 8 Preparation of Factor X

Solvent/detergent-treated prothrombin complex concentrate was applied toa column of copper-charged Chelating Sepharose Fast Flow in a solutioncontaining 0.5M sodium chloride. The column was washed with citratephosphate buffer pH6.5 containing 0.5M sodium chloride. The factor Xprotein was then eluted with citrate phosphate buffer pH7.0 containing0.1M sodium chloride. The protein eluted in the first 1.2 gel bedvolumes was discarded. The factor X protein eluted in the next 3.3 gelbed volumes was collected. The conductivity of this factor X solutionwas reduced to 10-18 mS/cm and the pH reduced to 6.0, then the solutionwas applied to a column of DEAE Sepharose Fast Flow anion exchange gel.The column was then washed with citrate buffer containing 0.1M sodiumchloride, then with citrate phosphate buffer containing 0.15M sodiumchloride. Purified factor X was then eluted with citrate phosphatebuffer pH7 containing 0.31M sodium chloride. The factor X was passedthrough a Planova 15 nm virus-retentive filter, formulated with 1% w/wsucrose, diluted to a target potency of 130 iu factor X per mL andfreeze-dried. The freeze-dried factor X vials were sealed under vacuum,then heated at 80° C. for 72 hours in a hot-air oven. Results are shownin Table 8.

TABLE 8 Purification of factor X Specific Factor X, Factor II, inactivity, iu/mg Stage iu/mL per 100 iuFX of protein Yield, % S/D PCC41.9 110 3.5 100 Metal chelate 12.7 0.88 109 24 eluate Ion-exchange 2510.42 135 94 eluate Virus filtrate 227 0.47 130 99 Freeze dried 130 0.51131 87 factor X Heat-treated 122 0.50 115 94 factor X

EXAMPLE 9 Preparation of Activated Factor X

Factor X was prepared as described in Example 8 above and diluted toapproximately 10 iu/ml. The solution was then mixed with Sepharose gelto which had been coupled the factor X activating protein of Russell'sViper Venom (RVVX). Activation occurred as the factor X was incubatedwith the RVV-X Sepharose for up to 3 hours and the activated factor Xsolution was then separated from the RVV-X Sepharose. Activation wasdemonstrated by the appearance of the factor Xa band on SDS-PAGE rununder reducing conditions. Activity was measured using a factor X assayin which the normal activator had been omitted and replaced with assaybuffer. One unit of activated factor X (FXa) was defined as the activityafter full activation of one international unit of factor X. Thepresence of native, nonactivated factor X was measured by performing theroutine factor X assay containing activator. Results (shown in Table 9)confirmed that more than 90% of the maximum activation was achieved.

TABLE 9 Factor X activity Factor X activity without assay with assayTest Sample activator, iu/mL activator, iu/mL Factor X start Notmeasured 9.2 material Factor X gel 5.9 6.3 supernatant

EXAMPLE 10 Use of Activated Factor X to By-Pass Factor VIII ClottingFunction

Activated factor X was prepared as described in Example 9 above.Different concentrations were added to a modified APTT assay system inwhich the normal plasma substrate had been replaced with factorVIII-deficient plasma. Results are shown in Table 10. Addition of bufferor inactivated factor X (prepared as described in Example 11) failed tocorrect the prolonged clotting times. Addition of activated factor Xachieved normal clotting parameters when added at a concentration ofbetween 0.02 and 0.05 u/mL.

TABLE 10 Clotting times in factor VIII-deficient substrate plasmaClotting Time, seconds Sample Reference plasma 47.4 Buffer 121.8Inactivated factor X, 20 u/mL* 105.5 5 mM Calcium chloride 121.8Activated factor X: 0.0005 u/uL 108.2  0.001 u/mL 107.7  0.025 u/mL 94.2 0.005 u/mL 86.9  0.01 u/mL 72.0  0.02 u/mL 59.2  0.05 u/mL 39.7   0.1u/mL 26 *the amount of inactive factor X obtained from 20 iu/mL offactor X

EXAMPLE 11 Preparation of Inactivated Factor X

Factor X was prepared by purification of solvent/detergent-treatedprothrombin complex concentrate by copper-charged metal chelate chelatechromatography followed by ion-exchange chromatography as described inpreceding examples. Factor X at 100 iu/mL and 10 iu/mL was theninactivated by incubation at 60° C. for up to 20 hours. The inactivationof factor X was measured by clotting time assay (Table 11). A clottingtime of 40 seconds approximates to 0.07 iu factor X/mL. These resultsshow that much less than 0.07 iu/mL of activity remained after 21 hours.Thus, substantially more than 99.9% inactivation was achieved.

TABLE 11 Clotting times (seconds) for Factor X activity duringinactivation process Factor X starting Heating Time, hours Concentration0 h 0.5 2 h 4 h 21 h 100 iu/mL 25.9 32.7 60 89.8  10 iu/mL 31.0 32.135.3 50.8 >150

EXAMPLE 12 Use of Inactivated Factor X to Inhibit Coagulation

Inactivated factor X (FX-IN) was prepared by heating factor X product at60° C. for 20 hours. Activated factor X (FXa) was diluted and mixed withdifferent concentrations of inactivated factor X, before adding tosubstrate in the Thrombin Generation Assay (TGA). Results are shown inTable 12. This shows that the coagulant effect of 0.05 u/ml FXa can benegated by the presence of between 5 and 10 units of inactivated factorX, equivalent to approximately 10-20 fold excess of inactive factor Xover activated factor X.

TABLE 12 Inhibition of coagulation by inactivated factor X Mean lag timeSample (minutes) Area under curve Velocity index Buffer 17.0 2051 1.6FXa, 0.1 u/mL^([1]) 9.0 3535 7.6 FXa, 0.05 u/mL^([1]) + 12.5 3290 5.1FX-IN 0.5 u/mL^([2]) FX-IN 2.5 u/mL^([2]) 14.0 3016 6.2 FX-IN 5.0u/mL^([2]) 13.5 2884 5.6 FX-IN 010 u/mL^([2]) 23.0 1544 1.4 FX-IN 25u/mL^([2]) 22.5 599 0.7 FX-IN 50 u/mL^([2]) 54.5 186 0.3 ^([1])1 unit ofFXa is defined as the amount obtained from 1 international unit offactor X after full activation. ^([2])1 unit of inactive factor X isdefined as the amount obtained from 1 international unit of functionalfactor X

EXAMPLE 13 Absence of Thrombogenicity in Factor X Preparation

Factor X was prepared as described in Example 8. The product was testedusing an in vivo rabbit stasis thrombogenicity model. The effect ofFactor X at three doses (100, 200 and 400 iu/kg body weight) wascompared with the effect of a physiological saline negative control. Athrombin positive control (50 iu/kg body weight) was included todemonstrate that the test system could detect a thrombogenic challenge.Products were infused into a marginal ear vein and allowed to enter thecirculation for 30 seconds. A section of jugular vein was then isolatedby ligature and the amount of thrombin formation was evaluated 15minutes later. Results showed no statistical difference between therabbits receiving factor X doses and rabbits receiving the salinenegative control.

The invention claimed is:
 1. A method of reversing the effect of ananticoagulant in a patient, the method comprising treating the patientwith a composition comprising a therapeutically effective amount offactor X or factor Xa and one or more compounds selected from the groupconsisting of pharmaceutically acceptable diluents, excipients, andstabilizers, treating the patient with a composition comprising atherapeutically effective amount of factor X or factor Xa furthercomprising preparing the factor X or factor Xa by a process comprisinga) adsorbing a starting material comprising factor X and prothrombinonto an immobilised metal ion affinity chromatography substrate; b)eluting selected adsorbed proteins from the substrate; c) monitoring theeluate for commencement of elution of prothrombin and factor X,discarding a first portion of the eluate and collecting a subsequentsecond portion of the eluate enriched in factor X; and d) reducing thelevel of residual prothrombin in the factor X obtained in step c) byanion exchange chromatography.
 2. A method according to claim 1, theprocess further comprising (i) solvent detergent treatment of a startingmaterial comprising factor X and prothrombin; ii) adsorbing the solventdetergent treated starting material onto an immobilised metal ionaffinity chromatography substrate; iii) eluting adsorbed proteins fromthe substrate; iv) monitoring the eluate for commencement of elution ofprothrombin/factor X, discarding a first portion of the eluate andcollecting a subsequent second portion of the eluate enriched in factorX; v) carrying out anion exchange chromatography to reduce the level ofresidual prothrombin in the second portion of the eluate; vi) filteringthe product of step v) through a virus-retentive filter; vii) freezedrying the filtered product of step vi), optionally in the presence ofone or more stabilizers; and viii) subjecting the freeze dried productto a virus reduction/inactivation heat treatment step.
 3. A methodaccording to claim 1, the process further comprising an activation stepto activate the factor X.
 4. A method according to claim 3, the factor Xbeing activated by a divalent metal ion alone or in combination with avenom.